SUMMARY Toxoplasma gondii is an opportunistic protozoan parasite that poses a significant risk to AIDS/HIV patients. Current treatments for toxoplasmosis are ineffective against the long-lived encysted stage of T. gondii. Thus, a constant stream of new drugs based on the identification of novel drug targets is required to offer long-term protection against cyst reactivation. Toxoplasma is an obligate intracellular parasite that multiplies in the cytoplasm of mammalian cells within a parasitophorous vacuole that does not fuse with any host organelles. Differentiated parasites into cyst forms (bradyzoites) are surrounded by a thick cyst wall within mammalian cells. How these parasites acquire their nutrients from the host cell has been an intriguing question in the field. We previously demonstrated that one mechanism developed by replicating tachyzoites to gain access to lipids is through the recruitment and sequestration of host nutrient-filled organelles into the parasitophorous vacuole. For example, these parasites retrieve cholesterol and sphingolipids from host endocytic organelles and Golgi- derived vesicles internalized into the vacuole, respectively. We provide new evidence that the cyst forms also salvage cholesterol. Present in all mammalian cells, lipid droplets represent an opportune source of neutral lipids for a lipid scavenger such as Toxoplasma. We show that both tachyzoites and bradyzoites rely on host lipid droplets for growth in vitro and infectivity in animals. Exogenous addition of fatty acids to the medium stimulate lipid droplet biogenesis in tachyzoites and bradyzoites, indicating that these parasites are capable of scavenging fatty acids that they use to esterify neutral lipids for storage in their own LD. The overall goal of our proposal is to unravel the mechanisms of host neutral lipid uptake and storage in tachyzoites and bradyzoites by identifying parasite effectors involved in these pathways. Specific Aim 1 will identify the parasite effectors involved in host lipid droplet recognition and sequestration into the parasitophorous vacuole. Specific Aim 2 will investigate the source of cholesterol for bradyzoites and the importance of enzymes synthesizing neutral lipids for chronic infection. Specific Aim 3 will investigate the content and biological properties of the lipid droplets in tachyzoites and bradyzoites. We will intend to open new avenues for therapeutic interventions based on restricting the parasite?s access to major host lipid sources and blocking lipid storage pathways in Toxoplasma.